RESEARCH ARTICLE 3519
Development 138, 3519-3531 (2011) doi:10.1242/dev.064006
© 2011. Published by The Company of Biologists Ltd
Ascl1 expression defines a subpopulation of lineagerestricted progenitors in the mammalian retina
Joseph A. Brzezinski, 4th1, Euiseok J. Kim2,*, Jane E. Johnson2 and Thomas A. Reh1,†
SUMMARY
The mechanisms of cell fate diversification in the retina are not fully understood. The seven principal cell types of the neural
retina derive from a population of multipotent progenitors during development. These progenitors give rise to multiple cell types
concurrently, suggesting that progenitors are a heterogeneous population. It is thought that differences in progenitor gene
expression are responsible for differences in progenitor competence (i.e. potential) and, subsequently, fate diversification. To
elucidate further the mechanisms of fate diversification, we assayed the expression of three transcription factors made by retinal
progenitors: Ascl1 (Mash1), Ngn2 (Neurog2) and Olig2. We observed that progenitors were heterogeneous, expressing every
possible combination of these transcription factors. To determine whether this progenitor heterogeneity correlated with different
cell fate outcomes, we conducted Ascl1- and Ngn2-inducible expression fate mapping using the CreERTM/LoxP system. We found
that these two factors gave rise to markedly different distributions of cells. The Ngn2 lineage comprised all cell types, but retinal
ganglion cells (RGCs) were exceedingly rare in the Ascl1 lineage. We next determined whether Ascl1 prevented RGC
development. Ascl1-null mice had normal numbers of RGCs and, interestingly, we observed that a subset of Ascl1+ cells could give
rise to cells expressing Math5 (Atoh7), a transcription factor required for RGC competence. Our results link progenitor
heterogeneity to different fate outcomes. We show that Ascl1 expression defines a competence-restricted progenitor lineage in
the retina, providing a new mechanism to explain fate diversification.
INTRODUCTION
The retina comprises seven principal cell types: rod and cone
photoreceptors, amacrine, bipolar and horizontal interneurons,
Müller glia and retinal ganglion cells (RGCs). Retroviral lineagetracing studies in rodents have shown that all of these cell types
derive from a common multipotent progenitor population (Turner
and Cepko, 1987; Turner et al., 1990), though fate-restricted
progenitors were not observed. Nonetheless, the time of permanent
cell cycle exit (‘birthdate’) for each cell type follows a stereotypical
pattern (Carter-Dawson and LaVail, 1979; La Vail et al., 1991;
Rapaport et al., 2004; Sidman, 1961; Young, 1985). This change in
production of the different cell types over the period of
retinogenesis has led several investigators to propose that the
competence (potential) of progenitors changes over time (Reh and
Kljavin, 1989; Watanabe and Raff, 1990), which probably depends
on the combination of genes expressed by progenitors (Livesey and
Cepko, 2001). Attempts to link directly heterogeneity in
progenitors to differences in competence have failed to identify
transcription factors that define restricted retinal progenitor
populations.
One family of transcription factors that has been shown to be
important in the regulation of cell fate is the basic helix-loop-helix
(bHLH) family. Three of these transcription factors, Olig2, Ngn2
(Neurog2) and Ascl1 (Mash1), are expressed in subpopulations of
1
Department of Biological Structure, University of Washington, Seattle, WA 98195,
USA. 2Department of Neuroscience, UT Southwestern Medical Center, Dallas,
TX 75390, USA.
*Present address: Neurobiology Section, Division of Biological Sciences, University of
California, San Diego, CA 92093, USA
†
Author for correspondence ([email protected])
Accepted 6 June 2011
progenitor cells throughout the nervous system, including the retina
(Guillemot and Joyner, 1993; Jasoni and Reh, 1996; Jasoni et al.,
1994; Lu et al., 2000; Nakamura et al., 2006; Shibasaki et al., 2007;
Sommer et al., 1996; Takebayashi et al., 2000). Previous studies
concluded that these factors are made by retinal progenitors;
however, no study has yet analyzed whether these factors are
expressed in overlapping or distinct progenitor populations, i.e.
whether they indeed define progenitor heterogeneity.
To address this, we first examined Olig2, Ascl1 and Ngn2
expression in progenitors. We found cells that expressed every
single, double and triple combination of these factors in the
developing retina, consistent with the hypothesis that these factors
define progenitor heterogeneity. To determine whether this
transcription factor heterogeneity corresponded to different fate
outcomes in the progenitors, we conducted Ascl1- and Ngn2inducible expression fate mapping using the CreERTM/LoxP system
(Metzger and Chambon, 2001; Metzger et al., 1995). We found that
Ascl1 and Ngn2 gave rise to very different distributions of retinal
cells. Ascl1-expressing cells can give rise to all major types of
retinal cells, though RGCs were not significantly represented.
Interestingly, we found that Ascl1+ cells could express the RGC
competence factor Math5 (J. A. Brzezinski, 4th, PhD thesis,
University of Michigan, 2005) (Mu et al., 2005; Yang et al., 2003),
but this subpopulation of cells did not adopt RGC fate. Together,
our data show that gene expression heterogeneity in retinal
progenitors leads to different fate choice outcomes: Ascl1
expression defines a competence-restricted lineage in the retina.
MATERIALS AND METHODS
Animals and tamoxifen administration
All animals were used in accordance with University of Washington and
UT Southwestern IACUC approved protocols. To trace the lineage of
Ascl1+ progenitors, we used the Ascl1CreERT2 knock-in mouse strain in
DEVELOPMENT
KEY WORDS: Development, Neurogenesis, Retina, Mouse
3520 RESEARCH ARTICLE
Immunohistochemistry and microscopy
Embryos, explants and P0 eyes were fixed for 1 hour in 2%
paraformaldehyde (PFA). Adult mice were transcardially perfused with 10
ml of 2% PFA. The eyes were then processed for cryosectioning.
Immunostaining was carried out using primary antibodies (below) and
appropriate fluorescent secondary antibodies and streptavidin conjugates
as previously described (Brzezinski et al., 2010).
The following primary antibodies were used: mouse anti-Ascl1 (1:100;
556604, BD Biosciences, San Jose, CA, USA), goat anti-Brn3a/b/c (panspecific) (1:50; sc6026, Santa Cruz Biotechnology, Santa Cruz, CA, USA),
rabbit anti-Brn3a (1:500; Eric Turner, Seattle Children’s Research Institute
and The University of Washington, Seattle, WA), rabbit anti-Brn3b (1:500;
Eric Turner) rabbit anti-calbindin D-28K (1:500; ab1778, Millipore,
Billerica, MA, USA), sheep anti-Chx10 (1:200; X1179P, Exalpha, Shirley,
MA, USA), mouse anti-Cre recombinase (1:250; mab3120, Millipore),
chicken anti-GFP (1:750; ab13970, Abcam, Cambridge, MA, USA), rabbit
anti-neurofilament-M (1:500; ab1987, Millipore), goat anti-Ngn2 (1:50;
sc19223, Santa Cruz), rabbit anti-Olig2 (1:250; ab33427, Abcam), goat
anti-Otx2-biotin (1:150; BAF1979, R&D Systems, Minneapolis, MN,
USA), rabbit anti-Pax6 (1:500; PRB-278P, Covance, Princeton, NJ, USA),
mouse anti-PKC (1:250; P5704, Sigma), goat anti-Sox2 (1:100; sc17320,
Santa Cruz) and rabbit anti-Sox9 (1:500; ab5535, Millipore).
Retrograde RGC labeling
RGC cell bodies were labeled by retrograde transport of biotinylated
dextrans (Farah and Easter, 2005). Crystals of 3000 weight lysine fixable
biotinylated dextran (Invitrogen) were liberally dissolved on Surgifoam
(Ethicon, Somerville, NJ, USA) soaked in 3% L-a-lysophosphatidylcholine
(Sigma). Eyes from recently euthanized lineage traced mice were
immediately placed on the dextran-coated Surgifoam, optic nerve remnant
down, and encased in 2% low-melt agarose/PBS. Eyes were placed in
HBSS+ (HBSS with 0.05 M HEPES and 6 mg/ml glucose) for 2 hours
under constant aeration. The Surgifoam and agarose were removed and the
eyes placed in fresh HBSS+ overnight under constant aeration. A hole was
made in the cornea and the eyes fixed for 3 hours in 2% PFA and processed
for histology (as above).
were cultured for 48 hours at 37°C and 5% CO2 with constant nutation. At
47 hours of culture, 1 µg EdU (5⬘-ethynyl-2⬘-deoxyuridine) (Invitrogen)
was added to the cultures and the explants rocked for an additional hour.
Explants were quickly dissociated in trypsin, pelleted, resuspended in TR
media, and plated on poly-D-lysine-coated glass coverslips. After 30
minutes, the media was removed and the cells fixed for 7 minutes with 2%
PFA. Cells were stained for DAPI and EdU using a Click-iT 555 Kit
according to the manufacturer’s instructions (Invitrogen).
EdU birthdating
A single intraperitoneal injection of 200 µg EdU was given to an E13.5
timed pregnant BL/6 female. Retinas were harvested from P22 animals,
fixed, cryopreserved and sectioned at 10 µm. Sections were stained for
calbindin, pan-Brn3 and DAPI as described above. Sections were stained
for EdU using a Click-iT 647 Kit (Invitrogen).
Cell counting and statistics
For the analysis of the overlap in expression of the progenitor transcription
factors, we manually counted Ascl1-GFP+, Ngn2+, Sox2+ and Olig2+
nuclei from sections of E14.5 and E18.5 Ascl1GFP/+ heterozygous and null
retinas. We counted from at least seven 600⫻ fields from two to four eyes
for each time point, representing ~4300 cells at E14.5 and ~6500 cells at
E18.5. Pan-Brn3+ RGCs were counted from at least five 400⫻ fields from
four eyes from E14.5 and E18.5 Ascl1 heterozygous and null retinas. This
represented ~1450 Brn3+ cells at E14.5 and ~1600 Brn3+ cells at E18.5.
Differences in cell populations were evaluated by two-tailed unpaired ttests. We counted Ascl1-GFP+, Math5-Cre+ and Brn3+ nuclei from at least
five 600⫻ fields from four eyes at E13.5 and E18.5, representing ~2100
and ~1250 cells, respectively. To determine the effects of Ascl1 loss on
proliferation in E18.5 explants, we counted DAPI+ and EdU+ nuclei from
17 or 18 200⫻ fields representing three heterozygous (~15,600 DAPI+
nuclei) and three Ascl1-null (~14,000 DAPI+ nuclei) mice, respectively.
Differences in the EdU+ fraction were evaluated using a two-tailed
unpaired t-test.
For the Ascl1 and Ngn2 lineage traces, cell types were characterized by
laminar position and unique morphology. The assignment of RGC versus
displaced amacrine cell was made by the presence or absence, respectively,
of pan-Brn3, Brn3a/b (in tandem), Neurofilament-M (NFM) or retrograde
biotinylated dextran uptake. When counting GFP+ cells, we grouped them
into ‘clumps’ based on their arrangement into radial units. Early born
neurons near a radial unit were included because some neurons tangentially
migrate a modest distance from their radial unit (Fekete et al., 1994; Reese
et al., 1999). We counted 1591 isolated cells and clumps (3115 cells) from
20 eyes for the Ascl1 lineage traced at E12.5/E13.5 and 2528 isolated cells
and clumps (4749 cells) from ten eyes for the Ascl1 lineage traced at
E17.5. We counted 1075 isolated cells and clumps (1254 cells) from ten
eyes for the Ngn2 lineage traced at E12.5/E13.5 and 827 isolated cells and
clumps (1088 cells) from four eyes for the E17.5 Ngn2 lineage trace (see
Table S1 in the supplementary material). Differences in fate distribution
were evaluated by 2 tests. Retroviral lineage data and adult cell
distributions were taken from Turner et al. (Turner et al., 1990) and Jeon
et al. (Jeon et al., 1998), respectively. Differences in clump sizes were
analyzed with the Mann-Whitney test. The binomial distribution was used
to determine the probability of observing a certain number of RGCs given
the number of cells sampled.
To determine the progenitor fraction in the E12.5/E13.5 Ascl1 lineage
at P0, all of the GFP+ cells observed in 30 sections from four eyes were
scored for the presence or absence of Sox9. The progenitor fraction from
E14.0 Ascl1 lineage assayed 23 hours after tamoxifen was calculated in the
same fashion (six sections, 212 GFP+ cells).
Retinal explant cultures
E17.5 Ascl1GFP/+ heterozygous and null retinas were collected in HBSS+
and placed RGC-layer up on 0.4 µm filter inserts (PICM03050, Milipore).
Explants were grown for 9 days at the gas/media interface at 37°C and 5%
CO2. Half of the media (TR media: Neuralbasal supplemented with 1⫻
penicillin/streptomycin, 1⫻ N2 supplement, 1⫻ L-glutamine and 1%
dialyzed FBS) (Invitrogen) was changed every other day. To measure
proliferation, E18.5 retinas from Ascl1GFP/+ heterozygous and null embryos
RESULTS
Ascl1 expression defines a subset of retinal
progenitor cells
To determine the relationships between the three bHLH
transcription factors known to be expressed in retinal progenitors,
we used antibodies to both Olig2 and Ngn2 as well as an
DEVELOPMENT
which CreERT2 replaces endogenous Ascl1 (Kim et al., 2011a; Kim et al.,
2011b). To track the expression of Ascl1, we used a previously
characterized mouse knock-in line in which the coding region of Ascl1 was
replaced by a nuclear localized GFP element (Leung et al., 2007). Both
Ascl1GFP/+ and Ascl1CreERT2 have all of the 5⬘ and 3⬘ regulatory elements
intact and GFP (or Cre) expression matches Ascl1 expression (Leung et al.,
2007). Heterozygous Ascl1GFP/+ mice were intercrossed to generate wildtype, heterozygous and Ascl1GFP/GFP-null mice, which did not survive past
birth. To track Math5 expression we used Math5Cre/+ knock-in mice (Yang
et al., 2003). For the Ngn2 lineage, the coding region of Ngn2 was replaced
by CreERTM (Ngn2CreERTM/+) (Ma and Wang, 2006; Zirlinger et al., 2002).
To monitor Cre activity, we used a dual reporter mouse line
(Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo, Jackson Stock 007576, i.e.
mTmG) that ubiquitously expressed membrane-localized tomato until
recombination, at which point it expressed membrane-localized GFP
(Muzumdar et al., 2007) from a CMV-b-actin enhancer/promoter element
inserted into the ROSA26 locus. To induce recombination, pregnant mice
were given 2 mg tamoxifen (T5648, Sigma, St Louis, MO, USA; 10 mg/ml
in corn oil) intraperitoneally at both embryonic day (E) 12.5 and E13.5, at
both E14.5 and E15.5, or at E17.5. The eyes from these mice were
collected for analysis at postnatal day (P) 0 or at P19-22. For short-term
analysis, E14.0 mice were given 2 mg of tamoxifen and collected 12 or 23
hours later.
Development 138 (16)
Ascl1 in restricted progenitors
RESEARCH ARTICLE 3521
Ascl1GFP/+ line of mice (Leung et al., 2007). Ascl1-GFP expression
in heterozygous knock-in mice was consistent with earlier reports
on Ascl1 expression in the mouse retina (Brown et al., 1998;
Guillemot and Joyner, 1993; Hufnagel et al., 2010). We observed
Ascl1-GFP expression in nuclei in the progenitor zone of the
central retina at E12.5 (Fig. 1A). At E13.5, Ascl1-GFP expression
had spread towards, but not yet reached, the far periphery (Fig.
1B). At E14.5, Ascl1-GFP extended to the far peripheral retina and
GFP+ nuclei were absent from the ganglion cell layer (GCL) (Fig.
1C-E). The majority of Ascl1-GFP cells co-expressed Sox2, a panprogenitor marker (71.9±4.12% s.d.) (Fig. 1C-E, Fig. 2D-E), but
only a small fraction of the Sox2 progenitor population made
Ascl1-GFP (25.6±3.99% s.d.) (Fig. 1C-E, Fig. 2E). The fraction
(~28%) of the Ascl1-GFP+ cells that did not express Sox2
probably represents transient persistence of GFP in postmitotic
cells derived from Ascl1+ progenitors; however, we cannot exclude
the possibility that some postmitotic cells transiently express Ascl1.
Similarly, only a subset of Sox2+ and Sox9+ progenitors expressed
Olig2 (14.1±3.57% s.d.) and Ngn2 (17.2±3.10% s.d.), respectively
(Fig. 2E).
To determine whether these transcription factors were expressed
in the same retinal progenitor cells, we compared the expression of
Ascl1-GFP with that of Ngn2 and Olig2. At E12.5 and E13.5, the
Ngn2 expression domain extended further peripherally than Ascl1GFP (Fig. 1A,B) and three populations of cells were present:
Ascl1-GFP+, Ngn2+ and cells that expressed both markers (Fig.
1A,B). At E14.5, we observed similar numbers of cells expressing
Olig2 or Ngn2, whereas about 50% more expressed Ascl1-GFP
(Fig. 1F-L, Fig. 2A). Interestingly, we found that all seven
molecularly distinct populations of cells existed in the retina at
E14.5 (Fig. 1F-L, Fig. 2C). The most abundant population
expressed solely Ascl1-GFP (19.3±4.49 s.d. cells per 100 m) (Fig.
2C). Some of these Ascl1-GFP-only cells are likely to be
postmitotic; however, this single labeled population was larger than
the Ascl1-GFP+, Sox2-negative population identified in parallel
experiments (7.75±1.58 s.d. cells per 100 m) (Fig. 2D), indicating
that most of the Ascl1-GFP-only cells are progenitors. The six
other population combinations were less common (about three to
nine cells per 100 m), but when pooled (31.2 cells per 100 m)
accounted for 50% more cells than Ascl1-GFP-only (Fig. 2C). Of
the cells that made Ascl1-GFP, it was equally likely that they coexpressed Ngn2 or Olig2 (Fig. 2B). A slightly smaller fraction of
the Ascl1-GFP+ population expressed all three markers
(20.1±4.75% s.d.) (Fig. 2B). Examining three transcription factors,
we found considerable progenitor heterogeneity in our snapshots
of retinal development at E14.5 and E18.5 (see Fig. S1 in the
supplementary material).
Ascl1+ progenitors form a fate-restricted lineage
The above results indicate that there is considerable heterogeneity
in the progenitor population with respect to bHLH gene expression.
One way to assess whether these progenitor populations are
equivalent is to determine whether they generate different lineages.
The lineages of Ngn2+ progenitor cells have already been analyzed
DEVELOPMENT
Fig. 1. Retinal progenitor transcription factor expression heterogeneity. (A,B)E12.5 (A) and E13.5 (B) Ascl1GFP/+ mouse retinas stained for
Ascl1-GFP (nuclear; green) and Ngn2 (red). Ascl1-GFP expression starts centrally and spreads towards the periphery with age (leading edge marked
with arrowheads). (C-E)E14.5 retinas stained for Sox2 (red) and Ascl1-GFP (green). A small number of Ascl1– GFP+ cells do not express Sox2
(arrowhead; enlarged in inset). (F-L)Ascl1-GFP (green), Olig2 (gray) and Ngn2 (red) staining of the E14.5 retina. Cells that express Ascl1-GFP only;
Olig2 only; Ngn2 only; Ascl1-GFP and Olig2; Ascl1-GFP and Ngn2; Olig2 and Ngn2; and all three transcription factors (arrows; enlarged in inset) are
observed. (M)Ascl1-GFP, Olig2 and Ngn2 expression in E14.5 Ascl1GFP/GFP-null retinas is similar to control (L). A co-expressing cell is enlarged in
inset. GCL, ganglion cell layer; L, lens; O, optic nerve head. Scale bars: 100m for A,B; 50m for C-M; 10m for higher magnification insets.
3522 RESEARCH ARTICLE
Development 138 (16)
(Ma and Wang, 2006), but this type of analysis has not been
reported for Ascl1+ progenitors. To determine the potential of
Ascl1+ cells we conducted a conditional expression fate-mapping
experiment utilizing Ascl1CreERT2/+ knock-in mice (Kim et al.,
2011a; Kim et al., 2011b), crossed to reporter mice that express
membrane localized GFP only after Cre-based recombination.
Tamoxifen was used to induce CreERT2 localization to the nucleus
and cause recombination. We injected tamoxifen at E12.5 and
E13.5 to ensure that the earliest cohort of Ascl1+ cells would be
represented in the lineage and to increase the total number of cells
that underwent recombination. We immunostained adult retinas
from reporter mice lacking Ascl1-Cre (Ascl1+/+) as a control and
did not detect any GFP+ cells. No GFP+ cells in Ascl1CreERT2/+
mice were detected if tamoxifen was not administered (data not
shown). We observed GFP+ cells distributed in all layers of the
retina in E12.5/E13.5 Ascl1CreERT2/+ lineage-traced mice (Fig. 3BM, see Fig. S2 in the supplementary material). GFP+ cells were
often found in small groups (clumps) or in isolation (Fig. 3B-M).
We observed GFP+ cells in peripheral retina, which strongly
suggests that recombination events occur at the leading edge of the
Ascl1 expression domain. However, we did not observe GFP+ cells
in the far peripheral retina, consistent with the lack of Ascl1
expression in that domain at E12.5 and E13.5, indicating that
CreER activity was neither protracted nor delayed.
When we assayed the cell types present in E12.5/E13.5 Ascl1
lineage-traced animals at 19-21 days of age, we found that all of
the principal cell types in the retina were well represented except
the ganglion cells (Fig. 3H-P, see Table S1 in the supplementary
material). Co-labeling for calbindin to mark horizontal and
amacrine cells (Fig. 3H,I), Pax6 to label amacrine (among other)
cells (Fig. 3J), Otx2 to mark photoreceptors and bipolar cells (see
Fig. S2 in the supplementary material), or Sox9 to label Müller glia
(data not shown) confirmed that all these cells types were present
in the Ascl1 lineage; however, the vast majority of the cells in the
Ascl1 lineage in the ganglion cell layer were displaced amacrine
cells as shown by: (1) the lack of labeling by ganglion cell-specific
markers pan-Brn3 (Brn3a, b, c), Brn3a/b (in tandem) or
Neurofilament-M (NFM) (Fig. 3B-D) (Nixon et al., 1989; Xiang et
al., 1995); (2) the inability to label the cells with retrograde uptake
of biotinylated dextran (Fig. 3E) (Farah and Easter, 2005); or (3)
DEVELOPMENT
Fig. 2. Transcription factor
expression in E14.5 Ascl1GFP/+
heterozygous and null
mouse retinas. (A)Plot
showing the number of cells
that express Ascl1-GFP, Ngn2
and Olig2 at E14.5. The
number of cells that express
each factor is similar in Ascl1
mutants (red) and
heterozygotes (black). (B)Plot
showing the percent of Ascl1GFP+ cells that co-express
Ngn2, Olig2 or both. Ascl1
mutants (red) have fewer Ascl1GFP+ cells that make Olig2
than controls (black) (unpaired
t-test, *P≤0.05). (C)Plot
showing the number of cells
that express each of seven
combinations of transcription
factors. Ascl1GFP/GFP-null mice
have a similar distribution of
cells. (D)Plot of Sox2+ and
Ascl1-GFP+ cell numbers at
E14.5. (E)Plot showing the
percentage of progenitors that
co-express Sox2 or Sox9. Error
bars for all panels represent
±s.d.
Ascl1 in restricted progenitors
RESEARCH ARTICLE 3523
the presence of AP2a staining, which specifically labels the bulk
of displaced amacrine cells (Fig. 3G) (Bassett et al., 2007). In our
analysis of the Ascl1 lineage, we identified only seven RGCs in the
3115 GFP+ cells sampled (see Table S1 in the supplementary
material). The paucity of ganglion cells in the Ascl1 lineage was
not due to lack of production at this age; we conducted a
birthdating analysis and found that 14% of cells that exited the cell
cycle at E13.5 adopted RGC fate (see Fig. S3, Table S2 in the
supplementary material). If we conservatively assume that RGCs
should be generated at their adult frequency (0.6%) (Jeon et al.,
1998) at the time of tamoxifen injection, then the probability of
observing at least seven RGCs in our sample size is 0.00181
(binomial distribution, see Table S3 in the supplementary material),
demonstrating that RGCs are significantly under-represented in the
DEVELOPMENT
Fig. 3. The E12.5/E13.5 Ascl1 lineage. Tamoxifen was administered at E12.5 and E13.5 and mice were examined as adults. (A)A control animal
lacking the Ascl1-Cre (Ascl1+/+) transgene. There is a fibrous GFP antibody-specific background in the ganglion cell layer (GCL; arrow) and inner
plexiform layer. (IPL). (B-P)Examples of Ascl1CreERT2/+ lineage-traced cells (isolated or in clumps) (green or gray) with cell-type specific labeling in A-J
(red). (B-J)Displaced amacrines (dA), inner nuclear layer (INL) amacrines (A), rods (R) and horizontal cells (H) are seen. (K-P)Examples of cones (C),
rods, bipolar cells (B), amacrine cells and Müller glia (M) seen in the Ascl1 lineage. (B-G)Cells in the GCL are labeled by pan-Brn3 (Brn3*), Brn3a/b,
Neurofilament-M (NFM) or with retrograde uptake of biotinylated dextran to label retinal ganglion cells (RGCs), and with AP2a to label displaced
amacrines (all in red). Nearly all GCL cells are label-negative displaced amacrine cells. (H-J)Horizontal and amacrine cells are co-labeled with
calbindin (red) or Pax6 (red). Arrowheads denote outer segments of photoreceptors. Scale bars: 50m for A-C,H-P; 25m for D-G; 10m for
insets.
3524 RESEARCH ARTICLE
Development 138 (16)
Ascl1 lineage. Only five of the ten animals we examined had GFP+
RGCs. Indeed, the RGC frequency (0.322±0.488% s.d.) was not
statistically different from zero (t-test, P>0.05) (see Fig. S4 in the
supplementary material). Thus, although an occasional ganglion
cell was seen in some Ascl1 lineage-traced mice, we can conclude
that the Ascl1 lineage effectively lacks RGCs.
In addition to the absence of ganglion cells in the Ascl1 lineage,
the relative numbers of cells in the E12.5/E13.5 Ascl1 lineage was
different from the normal adult mouse retina (Jeon et al., 1998).
Horizontals, cones and amacrines were over-represented whereas
rods were under-represented (see Fig. S4 in the supplementary
material). This early fate bias suggests that many Ascl1+ progenitors
exited the cell cycle near the time of tamoxifen administration. Many
of the later-born cell types (rods, bipolars, glia) were found in
multicellular clumps, suggesting that some Ascl1+ progenitors
labeled at E12.5/E13.5 divided multiple times before adopting a cell
fate. To examine this further, we gave mice tamoxifen at E12.5 and
E13.5 or at E14.5 and E15.5 and examined retinas at birth (P0). We
observed photoreceptors, amacrines, horizontal cells and progenitors
in the lineages traced from both time points but, again, no RGCs
were identified (see Fig. S5 in the supplementary material). About
one third (37.5±8.4% s.e.m.) of GFP+ cells in the E12.5/E13.5 trace
were Sox9+ progenitors at P0. We also gave tamoxifen at E14.0 and
examined the Ascl1 lineage 12 and 23 hours later. We observed few
GFP+ cells at 12 hours but numerous GFP+ cells at 23 hours (see
Fig. S6 in the supplementary material). Of these, half were Sox9+
progenitors (49.4±8.4% s.e.m.) whereas none co-expressed the RGC
marker pan-Brn3 (see Fig. S6 in the supplementary material). Taken
together, these data show that recombination and subsequent GFP
expression takes place quickly following tamoxifen treatment, that
Cre activity is not protracted, that recombination takes place in
proliferative Ascl1+ cells and that Ascl1+ cells can remain as
progenitors for several days.
We next examined the Ascl1 lineage after inducing Cre
recombination at E17.5. There were more GFP+ cells seen in
lineage-traced animals, correlating with a greater number of
Ascl1+cells at E17.5 compared with E13.5. GFP+ cells were seen
in isolation and in small clumps, both in the central and far
peripheral parts of the retina (Fig. 4B-L). Very few cells were
observed in the GCL and none of them co-expressed pan-Brn3 (see
Table S1 in the supplementary material). We observed few earlyborn cell types (cones, horizontals and displaced amacrines). These
occurrences were almost always observed in the peripheral retina,
where a small number of these cells are still being born on E17.5.
Most of the cells in the E17.5 Ascl1 lineage were rods and
amacrine cells (Fig. 4B-L). However, multicellular clumps that
contained postnatally generated cell types, such as Müller cells and
bipolar cells, were observed, indicating that some of the Ascl1+
DEVELOPMENT
Fig. 4. The E17.5 Ascl1 lineage. Tamoxifen was given at E17.5 and mice were examined as adults. (A)A control animal lacking Ascl1-Cre (Ascl1+/+)
has only background GFP staining (gray, arrow). (B-L)Examples of Ascl1CreERT2/+ lineage-traced cells (gray). Shown are examples of rods (R),
amacrines (A), bipolars (B), cones (C) and Müller glia (M). Arrowheads denote photoreceptor outer segments. Scale bars: 50m for A-L: 10m for
insets.
Ascl1 in restricted progenitors
RESEARCH ARTICLE 3525
progenitors continue to generate progeny for several days (Fig. 4BL). Taken together, our data show that Ascl1 expression defines a
population of competence-restricted progenitors in the retina.
Ascl1 itself does not inhibit RGC competence
Our lineage studies show that Ascl1+ progenitors do not become
RGCs. RGC development requires the transcription factor Math5
(Brown et al., 2001; Wang et al., 2001). Math5 is expressed in
postmitotic cells and establishes RGC competence, but only a subset
of Math5+ cells adopts RGC fate (J. A. Brzezinski, 4th, PhD thesis,
University of Michigan, 2005) (Mu et al., 2005; Yang et al., 2003).
In principle, Ascl1 could act by preventing the expression of Math5
in the Ascl1 lineage, thereby inhibiting RGC fate. Alternatively,
Ascl1+ progenitors could give rise to Math5+ postmitotic cells and
this subpopulation, owing to its previous expression of Ascl1, would
be inhibited from RGC fate. To test these hypotheses, we crossed
Ascl1GFP/+ mice to Math5Cre/+ mice and looked for overlap of GFP
and Cre proteins, which both persist transiently after Ascl1 and
Math5 expression terminates. We examined mice at E13.5 (the peak
of RGC formation) and at E18.5 (the tail of RGC formation) for panBrn3 co-expression. At E13.5, 36% of the cells in the retina are panBrn3+ RGCs (see Fig. S3 in the supplementary material) and most
of the Math5-Cre+ cells co-expressed pan-Brn3 (65.7±8.99% s.d.)
(Fig. 5A-D,M,N). By contrast, we observed only one instance of an
Ascl1-GFP+ cell that co-expressed pan-Brn3 (Fig. 5E-H,M,N, see
Table S4 in the supplementary material). Nonetheless, we observed
that many Ascl1-GFP+ cells co-expressed Math5-Cre at this age
(31.0±11.1% s.d.) (Fig. 5A-D,M,N). The same was true at E18.5; by
DEVELOPMENT
Fig. 5. Loss of Ascl1 does not affect retinal ganglion cell (RGC) numbers. (A-H)E13.5 Ascl1GFP/+::Math5Cre/+ transheterozygous mice stained
for Ascl1-GFP (green), Math5-Cre (gray) and pan-Brn3 (red). (A-D)The central retinas of E13.5 animals have many cells that co-express Math5-Cre
and Brn3. Numerous Ascl1-GFP+ cells co-express Math5-Cre (arrows), but none co-expresses Ascl1-GFP and Brn3. (E-H)E13.5 peripheral retina
showing the leading edge of Math5-Cre (white arrowhead), Brn3 (red arrowhead) and Ascl1-GFP (green arrowhead) expression. Only one cell made
Ascl1-GFP, Math5-Cre and Brn3 (arrows). (I-L)Pan-Brn3 stains of E14.5 (I,J) and E18.5 (K,L) Ascl1GFP/+ heterozygous (I,K) and null (J,L) retinas. C,
central; GCL, ganglion cell layer; P, peripheral. Scale bars: 50m for A-L; 10m for insets. (M)Plot of Ascl1-GFP, Math5-Cre and Brn3 cell counts at
E13.5 (black) and E18.5 (red). (N)Plot of overlap between Ascl1-GFP and Math5-Cre at E13.5 (black) and E18.5 (red). (O)Plot showing the number
of Brn3+ RGCs at E14.5 and E18.5 for Ascl1GFP/+ heterozygotes (black) and null (red) retinas. Error bars represent ±s.d. for all panels.
3526 RESEARCH ARTICLE
Development 138 (16)
this age, most Math5-Cre+ cells co-expressed Ascl1-GFP
(59.11±17.98% s.d.) (Fig. 5M,N, see Fig. S7 in the supplementary
material), but we did not detect Ascl1-GFP+/Brn3+ cells.
The fact that Ascl1+ progenitor cells can give rise to Math5expressing cells, but not Brn3+ ganglion cells suggests that prior
(or concurrent) Ascl1 expression prevents Math5+ cells from
adopting RGC fate. If Ascl1 prevents Brn3 expression in the
progeny of these progenitors, then the loss of Ascl1 should cause
an increase in RGC number. To test this hypothesis, we examined
the number of pan-Brn3+ RGCs at both E14.5 and E18.5 in
Ascl1GFP/+ heterozygous and null retinas and found no differences
at either age (Fig. 5I-L,O). The absence of an effect on ganglion
cell fate in the Ascl1GFP/GFP retinas might be due to compensation
by Ngn2 or Olig2. However, when we analyzed Ascl1GFP/GFP
retinas for the expression of Ngn2 and Olig2, we found that the
number of Ngn2+ or Olig2+ cells in Ascl1-null mice was not
significantly different compared with heterozygous mice at E14.5
(Fig. 1M, Fig. 2A,C). The only significant difference we observed
was that a slightly smaller fraction of Ascl1-GFP+ cells coexpressed Olig2 in the absence of Ascl1 (Fig. 2B). We also
examined Ascl1GFP/GFP retinas at E18.5. At this age, all populations
of progenitors (Sox2+, Ascl1-GFP+, Ngn2+ and Olig2+) were
modestly decreased (~15-30%) in Ascl1 mutants (see Fig. S1 in the
supplementary material). Thus, Ascl1 is not required for the
repression of the RGC fate in this lineage and neither compensation
nor cross-regulation by Ngn2 or Olig2 can explain this result.
Ascl1+ and Ngn2+ progenitors give rise to distinct
lineages
Previous conditional expression fate-mapping experiments
demonstrated that Ngn2+ progenitors can give rise to all retinal
cell types, including RGCs (Ma and Wang, 2006). As Ascl1 and
DEVELOPMENT
Fig. 6. Ngn2 lineage traces.
Tamoxifen was given at E12.5
and E13.5 or at E17.5 and
mice were examined as adults.
(A-J)E12.5/E13.5 Ngn2CreERTM/+
lineage traces (green) costained with pan-Brn3 (Brn3*),
Brn3a/b, or by retrograde
uptake of biotinylated dextran
to mark retinal ganglion cells
(RGCs; red). (A)Control mice
lacking Ngn2-Cre (Ngn2+/+)
have only fibrous GFP
background (arrow). (B-J)Ngn2
lineage-traced RGCs (G),
horizontals (H), cones (C) and
amacrines (A) are shown.
(F)Displaced amacrines (dA) are
marked with AP2a staining
(red) in the ganglion cell layer
(GCL). (K-N)E17.5 Ngn2
lineage trace showing rods (R),
amacrines (A), displaced
amacrines (dA) and cones (C).
Arrowheads mark
photoreceptor outer segments.
Scale bars: 50m for AN;
10m for insets.
Ascl1 in restricted progenitors
RESEARCH ARTICLE 3527
Ngn2 have partially overlapping domains at several time points
(Figs 1, 2, see Fig. S1 in the supplementary material), we tested
whether differences in expression of these factors in progenitors
leads to distinct lineages. We carried out lineage analysis of
Ngn2CreERTM/+ mice after administering tamoxifen at E12.5/E13.5
and at E17.5. Adult control mice without Ngn2-Cre (Ngn2+/+)
and Ngn2CreERTM/+ mice that did not receive tamoxifen lacked
GFP+ cells (Fig. 6A) (data not shown). Ngn2CreERTM/+ mice given
tamoxifen at E12.5/E13.5 had GFP+ cells in all layers of the
retina and were typically seen in isolation or in clumps of two to
three cells spanning the central and far peripheral retina (Fig.
6B-J). Most abundant in the E12.5/E13.5 Ngn2 lineage trace
were cones, horizontals and amacrine cells (Fig. 6F-J). There
were few rods, bipolars or Müller glia from this trace, which
contrasts significantly with the Ascl1 lineage trace (2 test,
P<0.001) (Fig. 7A, see Table S1 in the supplementary material).
GFP+ RGCs were present in every Ngn2 lineage-traced animal
we examined (Fig. 6B-E), and though not numerous, RGCs were
more frequently observed in the Ngn2 lineage (1.95±0.92 s.d.)
versus the Ascl1 lineage (0.322±0.488% s.d.) (Mann-Whitney,
P0.001, Fig. 7A, see Fig. S4 in the supplementary material).
Although the Ngn2 lineage was strongly biased towards cell
fates born around the time of tamoxifen administration, RGCs
formed only a small, though significant (t-test, P<0.01), fraction
of the Ngn2 lineage.
In the E17.5 Ngn2 lineage trace, most of the cells we observed
were rods, but a few early-born cell types were seen (e.g. RGCs
and cones), primarily in peripheral parts of the retina (Fig. 6K-N).
In contrast to the Ascl1 lineage, the latest generated cell types,
bipolars and Müller glia, were nearly absent from the E17.5 Ngn2
lineage (2 test, P<0.001) (Fig. 7B, see Table S1 in the
supplementary material). As before, the E17.5 Ngn2 lineage
distribution is highly biased towards the cell types born at the time
of tamoxifen administration. Together, our data show that Ascl1
and Ngn2 lineages are distinct. First, RGCs are significantly more
abundant in the Ngn2 lineage. Second, the Ngn2 lineage is more
heavily biased towards cell types that exit the cell cycle shortly
after tamoxifen treatment.
The differences in fate distributions suggested differential
proliferative ability of the Ascl1+ and Ngn2+ progenitor
populations. We counted the number of cells in clumps traced from
both time points as an indirect measure of the proliferative ability
of these progenitors. We plotted the frequency of clump sizes and
observed a Pareto-like (L-shaped) distribution for both lineages at
DEVELOPMENT
Fig. 7. Fate distribution and clump
sizes in the Ascl1 and Ngn2 lineages.
(A)Plot of the fate distributions of
E12.5/E13.5 Ascl1 (black) and Ngn2 (red)
lineages. The Ascl1 lineage and Ngn2
lineages have significantly different
distributions (2, P<0.001); the Ascl1
lineage nearly lacks retinal ganglion cells
(RGCs) and contains more late-born cell
fates. (B)Distribution of E17.5 Ascl1
(black) and Ngn2 (red) lineages.
Compared with the Ngn2 lineage, the
Ascl1 lineage has more late-born cell
types (2, P<0.001). G, RGC; H,
horizontal; C, cone; dA, displaced
amacrine; A, amacrine; R, rod; B, bipolar;
M, Müller glia. Error bars represent
±s.e.m. (C,D)Histogram showing clump
size frequencies in the E12.5/E13.5 (C)
and E17.5 (D) Ascl1 (black) and Ngn2
(red) lineages. The maximum clump size
is 15 cells for the Ascl1 lineage and nine
cells for the Ngn2 lineage at E12.5/13.5
(12 and seven cells, respectively, at
E17.5). (E)Plot showing the average size
of clumps in the Ascl1 and Ngn2
lineages. Ascl1 clumps are twice as large
as Ngn2 clumps (Mann-Whitney test,
**P<0.001). Error bars represent ±s.d.
both time points (Fig. 7C,D). The average clump size was about
twice as large in the Ascl1 lineage compared with the Ngn2 lineage
at both time points (Mann-Whitney test, P<0.001) (Fig. 7E). The
high frequency of one- and two-cell clumps in the Ngn2 lineage
(98.5%) suggests that Ngn2+ progenitors are typically in their last
cell cycle.
As noted above, all populations of E18.5 progenitors were
modestly decreased (~15-30%) in Ascl1GFP/GFP-null retinas (see
Fig. S1 in the supplementary material), consistent with a previous
study that suggested that Ascl1 non-autonomously maintains the
retinal progenitor pool (Nelson et al., 2009). We also found that
E18.5 Ascl1GFP/GFP-null explants cultured for 2 days in vitro (DIV)
had 50% fewer EdU+ (S-phase) nuclei (progenitors) at the end of
the culture period compared with heterozygous explants (see Fig.
S8 in the supplementary material). Lastly, we examined E17.5
retinal explants cultured for 9 DIV and found that although Ascl1null retinas were noticeably smaller, all seven principal retinal cell
types were present (data not shown, see Figs S1, S9 in the
supplementary material). Together these results suggest that Ascl1
is required for proliferation, which cannot be compensated for by
Olig2 or Ngn2.
DISCUSSION
Here, we report that retinal progenitors are heterogeneous in their
expression of the bHLH transcription factors Ascl1, Ngn2 and Olig2.
This progenitor heterogeneity is reflected in differences in fate; Ascl1
and Ngn2 lineages are distinct from each other. The Ascl1+
progenitor cells do not significantly generate RGCs, but can give rise
to the other six principal retinal cell fates. Ascl1 expression,
therefore, defines a subpopulation of competence-restricted
progenitors during retinal development. Interestingly, Ascl1 itself is
not required to restrict RGC competence, suggesting a mechanism
by which factors upstream of Ascl1 limit competence in the retina.
Ascl1 defines a competence-restricted retinal
lineage
Ascl1+ progenitors did not significantly generate RGCs at any time
point. Although we cannot formally rule out that a biologically
relevant, rare RGC subtype(s) derives from the Ascl1 lineage, our
data strongly argue against this possibility. First, Ascl1-GFP and
pan-Brn3 co-expression data suggests that this putative subtype
would have to be exceedingly rare during development (one cell or
fewer per retina) (binomial distribution, P<0.00001, see Table S5
in the supplementary material). Second, whereas Brn3a/b/c
expression might not label all ganglion cell subtypes (Badea and
Nathans, 2011), retrograde dextran uptake labels all RGCs;
nonetheless, we did not observe a significant number of Brn3+ or
dextran-labeled RGCs in the Ascl1 lineage.
Retroviral lineage-tracing studies have shown that all seven
retinal cell types derive from a common progenitor population
(Turner and Cepko, 1987; Turner et al., 1990). However,
throughout most of retinal development, several cell types are
being formed concurrently (La Vail et al., 1991; Rapaport et al.,
2004; Sidman, 1961; Young, 1985). This implies that retinal
progenitor cells form a heterogeneous population that expresses
different intrinsic factors and responds differentially to extrinsic
cues (Livesey and Cepko, 2001; Taylor and Reh, 1990). Previous
studies have demonstrated that Ascl1 is expressed in a subset of
retinal progenitors, and proposed that Ascl1 expression defines a
particular stage in the progenitor cells (Jasoni and Reh, 1996;
Jasoni et al., 1994). Our Ascl1 lineage-tracing experiment
confirmed this proposal, as Ascl1+ progenitors significantly
Development 138 (16)
generated all retinal cell types except for RGCs. This is the first
molecularly defined progenitor population that has competence to
form all but one cell type in the retina. Analogously, Ascl1+ cells
give rise to a competence-restricted lineage in the spinal cord
(Battiste et al., 2007), forebrain (Parras et al., 2002) and other
regions of the CNS (Kim et al., 2008).
Although lineage-restricted progenitors have not been previously
shown to exist in the mouse retina in vivo, it has been shown that
committed precursors exist in the fish retina. Raymond and
colleagues first demonstrated that many of the rod photoreceptors
in the teleost retina are generated through a committed rod
precursor (Raymond and Rivlin, 1987). More recently, immature
horizontal cells in the inner nuclear layer of the zebrafish retina
have been shown to undergo a mitotic division to generate two
horizontal cells (Godinho et al., 2007). In Ath5-GFP transgenic
zebrafish, GFP is made by cells in their terminal division such that
one daughter becomes a ganglion cell and the other adopts a
different neural fate (Poggi et al., 2005). This cell is somewhat
different from the committed horizontal and rod precursors, but
nevertheless suggests that, in fish, there is something unique about
progenitor cells in their last cell division. Our results suggest that
Ngn2 expression might mark mouse progenitors in their final
mitotic division since nearly all of the traced cells were found in
one- to two-cell clumps and because their cell fates were strongly
biased towards those born at the time of tamoxifen treatment.
However, we did not find any clear pattern to the types of progeny
generated in two-cell clumps in the Ngn2 or Ascl1 lineages (data
not shown) (Ma and Wang, 2006). This appears to be true in vitro
as well, where single isolated progenitors that undergo their final
mitotic division in culture do not show a bias towards generating
cells of the same identity (Gomes et al., 2011).
Our study demonstrates that the E12.5/E13.5 Ascl1 and Ngn2
lineages are quite distinct from each other and from retroviral
lineages traced at the same time points (see Figs S4, S10 in the
supplementary material) (Turner et al., 1990). However, these
lineage-tracing techniques are quite different from each other. First,
only progenitors can be infected and labeled by retroviruses, but it is
possible for Ascl1- and Ngn2-Cre to persist transiently and catalyze
recombination in newly postmitotic cells. Thus, the average clump
size in the Ascl1 and Ngn2 lineages should be lower than that in the
retroviral lineages, but the maximum clump size should be similar.
Second, although clumps seen in the E12.5/E13.5 Ascl1 and Ngn2
lineage traces were sparsely distributed, we cannot be certain they
were clones. Thus, we might be overestimating the number of
progeny that Ascl1+ and Ngn2+ cells generate.
A major difference between these three lineage traces was the
clump/clone size distribution. Whereas some retroviral clones were
larger than 50 cells (Turner et al., 1990), we did not observe any
clumps in the Ascl1 lineage that contained more than 15 cells and
no clumps in the Ngn2 lineage that contained more than nine cells
(see Fig. S10 in the supplementary material). Most of the clumps
in the Ascl1 and Ngn2 lineages contained one or two cells (82.4%
and 98.5%, respectively), but only a small fraction of retroviral
clones (~33%) contained one or two cells. These differences in the
clump/clone size distributions strongly suggest the following model
(Fig. 8). Retinal progenitors that do not express Ascl1 or Ngn2 can
undergo a large number of mitotic divisions, whereas progenitors
that express Ascl1 undergo few mitotic divisions and those that
express Ngn2 are in their last cell cycle. This model is consistent
with observations in other regions of the CNS, where it has been
proposed that Ngn2-expressing progenitors in the spinal cord are
in their last cell division (Helms et al., 2005) and that Ascl1
DEVELOPMENT
3528 RESEARCH ARTICLE
Fig. 8. Model based on lineage and expression data described in
this report. Progenitors (Pr, Sox2+) that express neither Ascl1 nor Ngn2
have the potential to generate large clones (>16 cells) and adopt all cell
fates. Ascl1+ progenitors (green) undergo fewer cell divisions (<16 cells)
and generate all cell fates except retinal ganglion cells (RGCs), thus
defining a competence-restricted lineage. Ngn2+ progenitors (red) can
adopt RGC and all other cell fates, but appear to be in their last cell
division. Many, or perhaps all, Ascl1+ progenitors in their last cell cycle
co-express Ngn2 (yellow) and subsequently adopt non-RGC fates.
overexpression can inhibit proliferation in certain contexts
(Alvarez-Rodriguez and Pons, 2009; Bertrand et al., 2002; Farah
et al., 2000).
The Ascl1, Ngn2 and retroviral lineages have distinct distributions
of other cell fates (see Fig. S4 in the supplementary material). These
distinctions, with the exception of RGCs, can be attributed to
differences in proliferation. Ascl1+ cells at E12.5/E13.5 primarily
adopted early neural fates, but some of the recombined cells
proliferate until at least P0 and generate later fates, such as rods,
bipolars and Müller glia. By contrast, the Ngn2 lineage fate
distribution is consistent with near total cell cycle exit at the time of
tamoxifen administration. The retroviral lineages from these same
ages contained large clones with, predominantly, cells born later in
development, such as rods, bipolars and amacrine cells (Turner et al.,
1990). Small retroviral clones (<16 cells) had more early-born fates,
but did not match the fate distribution of the Ascl1 or Ngn2 lineages
(see Table S1 in the supplementary material) (Turner et al., 1990).
Although the Ngn2 lineage cannot be an obligate subset of the Ascl1
lineage, our data suggest that these two lineages overlap such that
co-expressing cells are in their final cell division (Fig. 8).
We observed that Ngn2+ progenitors generated few RGCs.
Because RGCs and horizontal cells are similar in number and their
birthdates overlap extensively (Jeon et al., 1998; Rapaport et al.,
2004) these cell types should be similarly represented in the
E12.5/E13.5 Ngn2 lineage trace. However, RGC frequency was
considerably lower (~12-fold) than horizontal cells, indicating that
only a subset of RGCs come from Ngn2+ progenitors. This
suggests that Ngn2 lineage is largely RGC-competence restricted,
probably a result of considerable overlap with the Ascl1 lineage. It
is unclear whether Olig2+ progenitors are also competence
restricted. An initial examination of the E14.5 Olig2 lineage
revealed RGCs, amacrines and horizontal cells (Shibasaki et al.,
2007), suggesting that Olig2+ progenitors might be more similar to
Ngn2+ than to Ascl1+ progenitors in the retina.
Progenitor heterogeneity correlates with
different fate outcomes
Previous studies have demonstrated progenitor heterogeneity in the
retina. For example, progenitor cells from late retina differentiate
in response to increases in cAMP, whereas those from early
RESEARCH ARTICLE 3529
embryonic stages do not (Taylor and Reh, 1990). Analogously,
EGF and related EGFR ligands are mitogenic for late progenitors,
but not early ones (Anchan et al., 1991; Lillien and Cepko, 1992).
Progenitors are heterogeneous in their expression of the cyclindependent kinase inhibitors p57kip2 and p27kip1 (Dyer and Cepko,
2001). In our experiments, we found that all seven distinct single,
double and triple bHLH transcription factor combinations were
represented, along with Sox2+ progenitors that did not express
Ascl1, Ngn2 or Olig2. In single-cell gene expression-profiling
experiments, roughly similar fractions of progenitors expressed
Ascl1, Ngn2 or both factors (Trimarchi et al., 2008). Although we
have not analyzed the Olig2 lineage, at least two populations of
cells, Ascl1+ and Ngn2+, have different fate and proliferative
potentials. Thus, progenitor heterogeneity correlates with different
fate choice outcomes in the retina.
How do Ascl1, Ngn2 and Olig2 regulate cell diversity in the
retina? One model proposes that different bHLH transcription
factors bias retinal progenitor cells, or their postmitotic progeny, to
specific cell fates (Ohsawa and Kageyama, 2008); each factor (or
combination of factors) is instructive for a specific cell fate. This
seems unlikely for Ascl1 and Ngn2, as mice deficient in these
transcription factors are able to generate all of the seven principal
retinal cell types (Akagi et al., 2004; Hufnagel et al., 2010;
Skowronska-Krawczyk et al., 2009; Tomita et al., 2000).
Alternatively, these factors could bias progenitors towards neural
fates. Ascl1 and Ngn2 mutants have increased glia at the expense
of neurons, whereas overexpression of these factors drives neurons
(Akagi et al., 2004; Cai et al., 2000; Nelson et al., 2009; Tomita et
al., 2000; Tomita et al., 1996). A third possibility is that these
transcription factors differentially regulate progenitor cell cycle
dynamics; our lineage data show that Ascl1+ and Ngn2+ cells have
different proliferative and fate potentials. Perhaps a combination of
Ascl1 and Ngn2 reach a threshold and induce terminal
differentiation. Alternatively, Ascl1, but not Ngn2, might
autonomously promote proliferation of progenitors by activating
cell-cycle genes (Castro et al., 2011). Lastly, these transcription
factors might differentially activate cell non-autonomous signaling
mechanisms. Previously, we have shown that Ascl1-null mice
express lower levels of the Notch ligands Dll1 and Dll3 in
progenitors, resulting in diminished Notch signaling within the
retina (Nelson et al., 2009). One consequence of reduced Notch
signaling might be a progressive depletion of progenitors, a
phenotype that we observed in Ascl1-null mice. Notch ligand
reduction is not observed in Ngn2-deficient retinas, demonstrating
a difference between these transcription factors in sustaining Notch
signaling and the progenitor pool (Nelson et al., 2009; Ohsawa and
Kageyama, 2008).
Ascl1 is not required to restrict RGC competence
The bHLH transcription factor Math5 is required for RGC
formation (Brown et al., 2001; Wang et al., 2001). Math5 is
required for RGC competence, but only a small subset of Math5+
cells adopts RGC fate (J. A. Brzezinski, 4th, PhD thesis, University
of Michigan, 2005) (Mu et al., 2005; Yang et al., 2003). How are
Ascl1+ cells prevented from becoming RGCs? First, Ascl1 might
repress Math5 and prevent RGC competence. This is unlikely as
we saw numerous Ascl1+/Math5+ co-labeled cells and Math5
expression is unchanged in Ascl1-null mice (Hufnagel et al., 2010;
Nelson et al., 2009). Second, Ascl1 might restrict RGC competence
independently of Math5. This also seems unlikely because Ascl1
mutant mice had normal numbers of RGCs during development.
Ngn2+ cells, which can give rise to Math5+ cells and RGCs,
DEVELOPMENT
Ascl1 in restricted progenitors
engineered to co-express Ascl1 were still able to make Math5 and
become RGCs (Hufnagel et al., 2010). Also, overexpression of
Ascl1 in chicken retina did not change the number of RGCs (Mao
et al., 2009). Third, it is possible that Ascl1, Ngn2 and/or Olig2
function redundantly to repress RGC formation. This is consistent
with the paucity of RGCs in our Ascl1 and Ngn2 lineages traces
and with our Ascl1, Ngn2 and Olig2 expression data. In support of
this possibility, there is a modest increase in the number of RGCs
and amount of Math5 expression in Ascl1/Ngn2 double knockout
mice (Akagi et al., 2004). Lastly, another factor might restrict RGC
competence in Ascl1+ cells. Dicer conditional null retinas, which
cannot make microRNAs, lack Ascl1 expression (Georgi and Reh,
2010). However, unlike Ascl1-null mice, conditional Dicer-null
retinas have extra RGCs. This raises the possibility that the factors
that regulate Ascl1 expression also restrict RGC competence.
Together, our data suggest a developmental mechanism in which
factors upstream of, or redundant with, Ascl1 restrict RGC
competence to promote cell fate diversification in the retina.
Acknowledgements
We thank members of the Reh and Birmingham-McDonogh laboratories for
advice and technical assistance, especially Deepak Lamba, Sean Georgi and
Kristin Sternhagen; Tom Glaser and Lev Prasov for advice; and Branden Nelson
for valuable discussions and for his role in initiating this project. The
Ascl1CreERT2/+ mice were created under a collaborative arrangement with the
Alexandra Joyner (Sloan Kettering Institute, NY, USA) and Randall Reed (Johns
Hopkins University, MD, USA) laboratories. Randall Reed and Cheuk Leung
provided the Ascl1 genomic DNA containing the homology arms, E.J.K. and
J.E.J. generated the targeting construct, and Alexandra Joyner and Anamaria
Sudarov were responsible for targeting in ES cells and generating the targeted
Ascl1CreERT2 mouse line. We thank Russel Van Gelder, Randall Reed and David
Anderson for sharing animal reagents and Erick Turner for antibodies. We are
grateful for the use of the Lynn and Mike Garvey Cell Imaging Laboratory. This
work was supported by NIH R01 EY013475 to T.A.R. and R01 NS32817 to
J.E.J. J.A.B. was supported by NIH NRSA F32-EY19227. Deposited in PMC for
release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.064006/-/DC1
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